Angiogenesis, the formation of new blood vessels from preexisting vessels, promotes wound healing and fertility. However, angiogenesis also promotes tumor growth and metastasis, macular degeneration and inflammatory diseases (Carmeliet et al., 2003, 2003, Nat. Med. 9:653). Lymphangiogenesis, the development of new lymphatic vessels, also occurs during tumor development (Stacker et al., 2002, Nat. Rev. Cancer 2:573; Alitalo et al., 2005, Nature 438:946). Studies have shown that tumor metastasis to the sentinel lymph nodes occurs as a result of lymphangiogenesis within tumors (Karkkainen et al., 2002, Nat. Cell Biol. 4:E2). Metastasis to other loci, such as lung and bone, may also occur as a result of lymphangiogenesis.
The molecular mechanisms that regulate the lymphatic vessels growth have been poorly understood. Recently, several specific markers of the lymphatic vessels have been described and have facilitated the lymphangiogenesis study. Two members of the VEGF family, VEGF-C and VEGF-D, have been shown to regulate lymphangiogenesis by binding the receptors VEGFR-2 and VEGFR-3 on lymphatic endothelial cells (Joukov et al., 1996, EMBO J. 15:1751; Achen et al., 2000, Eur. J. Biochem. 267:2505; Achen and Stacker, 1998, Int. J. Exp. Pathol. 79:255). In addition, the homeodomain transcription factor Prox-1 has been shown to promote lymphatic endothelium specification (Wigle et. al., 1999, Cell 98:769) and podoplanin expression (Breiteneder-Geleff et. al., 1999, Am. J. Pathol. 154:385). In addition, the CD44 family member LYVE-1 was shown to be expressed selectively by lymphatic endothelium (Banerji et al., 1999, J. Cell Biol. 144:789; and Prevo et al., 2001, J. Biol. Chem. 276:19420). LYVE1 is expressed on the cell surface as a 60-kD protein, which is reduced to approximately 40 kD by glycosidase treatment. Expression of LYVE 1, but not of CD44, largely restricted to endothelial cells lining lymphatic vessels and splenic sinusoidal endothelial cells. Expression was undetectable on lymphocytes, hematopoietic cells, or vascular endothelial cells. LYVE-1 is a useful marker to determine the localization of lymphatic endothelium by immunohistochemistry in vivo and to characterize and purify LEC in vitro.
Lymphatic vessels differ from blood vessels in several ways. Large collecting lymphatic vessels contain vascular smooth muscle cells in their wall, as well as valves, which prevent the backflow of lymph. However, lymphatic capillaries, unlike typical blood capillaries, lack pericytes and continuous basal lamina and contain large inter-endothelial valve-like openings (Casley-Smith, 1980, Lymphology 13:177; Lohela et al., 2003, Thromb. Haemost. 90:167). Due to their greater permeability, lymphatic capillaries are more effective than blood capillaries in allowing tumor cells to pass.
Many tumors express VEGF-C and VEGF-D, growth factors that selectively regulate lymphangiogenesis (Kaipainen et al., 1995, Proc. Natl. Acad. Sci. 92:3566; Saaristo et al., 2000, Oncogene 19:6122). While there are three known vascular endothelial growth factor receptors, VEGFR-1, VEGFR-2, and VEGFR-3 (Saaristo et al., 2000), only one, VEGFR-3 is expressed predominantly on lymphatic vessel. VEGF-D has been demonstrated to bind VEGFR3 and to induce endothelial cell proliferation (Achen et al., 2000; Achen and Stacker, 1998). Importantly, tumor-associated macrophages can release VEGF-C, as in human cervical cancer (Schoppman et al., 2002, Am. J. Pathol. 161:947). In fact, recent studies showed that macrophage secretion of VEGF-C and VEGF-D induces lymphangiogenesis (Cursiefen et al., 2004, J. Clin. Invest. 113:1040), thereby inducing lymphangiogenesis in tumors (Makinen et al., 2001, EMBO J. 20:4762; Stacker et al., 2002).
The integrin family of cell adhesion proteins controls cell attachment to the extracellular matrix and promotes the survival, proliferation and motility of many cell types (Kim et al., 2000, J. Biol. Chem. 275:33920; Hood and Cheresh, 2002, Nat. Rev. Cancer 2:91). Integrins transduce intracellular signals that promote cell migration and cell survival. In contrast, inhibition of integrin-ligand interaction induces apoptosis (Kim et al., 2002, J. Clin. Invest. 110:933).
At least three integrins receptors for provisional matrix proteins (αvβ3, αvβ5 and α5β1) play important roles in angiogenesis (Stromblad and Cheresh, 1996, Trends Cell Biol. 6:462). The expression of integrins α4β1, αvβ3 and α5β1 may control angiogenesis; none are expressed by quiescent endothelium but are expressed in response to angiogenic growth factors. Once they are expressed, angiogenesis depends on each integrin as antagonists of each can block angiogenesis in vivo. Recent studies show that integrin α4β1 plays an important and unique role in angiogenesis by promoting endothelial cell-smooth muscle cell interactions during angiogenesis (Garmy-Susini et al., 2005, J. Clin. Invest. 115:1542).
Little is currently known about the roles of integrins in lymphangiogenesis, although integrin α9β1 has been shown to be required for lymphatic development (Huang et al. 2000, Mol. Cell. Biol. 20:5208). Integrin α9β1 has also been shown to bind directly VEGFC and VEGFD (Vlahakis et al., 2005, J. Biol. Chem. 280:4544) and to induce LEC attachment and migration. Thus there remains a need in the art for the identification of the roles of integrins in the regulation of lymphangiogenesis in cancer.